Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

ABSTRACTThe anaerobic gastrointestinal pathogenClostridium difficilemust form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by whichC. difficileinitiates and controls the early stages of sporulation inC. difficileare not highly conserved in otherClostridiumorBacillusspecies. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation inC. difficile. These permeases are known to positively affect sporulation inBacillusspecies through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases inC. difficileresulted in the earlier expression of early sporulation genes and increased sporulationin vitro. Furthermore, disruption ofoppandappresulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model ofC. difficileinfection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence inC. difficilethan inBacillus subtilisand suggest that nutrient availability plays a significant role in pathogenesis and sporulationin vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation inC. difficile.

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MBX-500, a Hybrid Antibiotic withIn VitroandIn VivoEfficacy against Toxigenic Clostridium difficile

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00508-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4786-4792 ◽

Cited By ~ 16

Author(s):

Michelle M. Butler ◽

Dean L. Shinabarger ◽

Diane M. Citron ◽

Ciarán P. Kelly ◽

Sofya Dvoskin ◽

...

Keyword(s):

Weight Gain ◽

Clostridium Difficile ◽

Severe Disease ◽

Normal Weight ◽

New Drugs ◽

Hamster Model ◽

Resistance Development ◽

Content Type

ABSTRACTClostridium difficileinfection (CDI) causes moderate to severe disease, resulting in diarrhea and pseudomembranous colitis. CDI is difficult to treat due to production of inflammation-inducing toxins, resistance development, and high probability of recurrence. Only two antibiotics are approved for the treatment of CDI, and the pipeline for therapeutic agents contains few new drugs. MBX-500 is a hybrid antibacterial, composed of an anilinouracil DNA polymerase inhibitor linked to a fluoroquinolone DNA gyrase/topoisomerase inhibitor, with potential as a new therapeutic for CDI treatment. Since MBX-500 inhibits three bacterial targets, it has been previously shown to be minimally susceptible to resistance development. In the present study, thein vitroandin vivoefficacies of MBX-500 were explored against the Gram-positive anaerobe,C. difficile. MBX-500 displayed potency across nearly 50 isolates, including those of the fluoroquinolone-resistant, toxin-overproducing NAP1/027 ribotype, performing as well as comparator antibiotics vancomycin and metronidazole. Furthermore, MBX-500 was a narrow-spectrum agent, displaying poor activity against many other gut anaerobes. MBX-500 was active in acute and recurrent infections in a toxigenic hamster model of CDI, exhibiting full protection against acute infections and prevention of recurrence in 70% of the animals. Hamsters treated with MBX-500 displayed significantly greater weight gain than did those treated with vancomycin. Finally, MBX-500 was efficacious in a murine model of CDI, again demonstrating a fully protective effect and permitting near-normal weight gain in the treated animals. These selective anti-CDI features support the further development of MBX 500 for the treatment of CDI.

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The HtrA-Like Protease CD3284 Modulates Virulence of Clostridium difficile

Infection and Immunity ◽

10.1128/iai.02336-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4222-4232 ◽

Cited By ~ 14

Author(s):

Dennis Bakker ◽

Anthony M. Buckley ◽

Anne de Jong ◽

Vincent J. C. van Winden ◽

Joost P. A. Verhoeks ◽

...

Keyword(s):

Clostridium Difficile ◽

Virulence Factors ◽

Pathogenic Bacteria ◽

Microarray Data Analysis ◽

Toxin A ◽

Hamster Model ◽

Golden Syrian Hamster ◽

Content Type

ABSTRACTIn the past decade,Clostridium difficilehas emerged as an important gut pathogen. Symptoms ofC. difficileinfection range from mild diarrhea to pseudomembranous colitis. Besides the two main virulence factors toxin A and toxin B, other virulence factors are likely to play a role in the pathogenesis of the disease. In other Gram-positive and Gram-negative pathogenic bacteria, conserved high-temperature requirement A (HtrA)-like proteases have been shown to have a role in protein homeostasis and quality control. This affects the functionality of virulence factors and the resistance of bacteria to (host-induced) environmental stresses. We found that theC. difficile630 genome encodes a single HtrA-like protease (CD3284; HtrA) and have analyzed its rolein vivoandin vitrothrough the creation of an isogenic ClosTron-basedhtrAmutant ofC. difficilestrain 630Δerm(wild type). In contrast to the attenuated phenotype seen withhtrAdeletion in other pathogens, this mutant showed enhanced virulence in the Golden Syrian hamster model of acuteC. difficileinfection. Microarray data analysis showed a pleiotropic effect ofhtrAon the transcriptome ofC. difficile, including upregulation of the toxin A gene. In addition,the htrAmutant showed reduced spore formation and adherence to colonic cells. Together, our data show thathtrAcan modulate virulence inC. difficile.

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The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

Infection and Immunity ◽

10.1128/iai.00735-16 ◽

2016 ◽

Vol 84 (12) ◽

pp. 3434-3444 ◽

Cited By ~ 17

Author(s):

Kevin O. Childress ◽

Adrianne N. Edwards ◽

Kathryn L. Nawrocki ◽

Sarah E. Anderson ◽

Emily C. Woods ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Clostridium Difficile ◽

Molecular Mechanisms ◽

Toxin Production ◽

Negative Regulator ◽

Hamster Model ◽

Content Type ◽

Sporulation Initiation

The formation of spores is critical for the survival ofClostridium difficileoutside the host gastrointestinal tract. Persistence ofC. difficilespores greatly contributes to the spread ofC. difficileinfection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation toC. difficilepathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved inC. difficileand few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion ofCD1492resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in theCD1492mutant. Deletion of CD1492 also resulted in decreased toxin productionin vitroand in decreased virulence in the hamster model of CDI. Further, theCD1492mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lowersigDandrstAtranscription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence thatC. difficilesporulation is regulated differently from that of other endospore-forming species.

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Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

Infection and Immunity ◽

10.1128/iai.02316-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 502-513 ◽

Cited By ~ 20

Author(s):

Shan Li ◽

Lianfa Shi ◽

Zhiyong Yang ◽

Yongrong Zhang ◽

Gregorio Perez-Cordon ◽

...

Keyword(s):

Clostridium Difficile ◽

Cysteine Protease ◽

Lethal Dose ◽

Enzymatic Activities ◽

Target Cells ◽

Content Type ◽

Cytopathic Effects

TcdB is one of the key virulence factors ofClostridium difficilethat is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (VHH) library raised against TcdB, we identified two VHH antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB inin vivotoxicity using a sensitive systemic challenge model in mice. Consistent with thesein vitroresults, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.

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Pharmacological, Toxicological, and Dose Range Assessment of OG716, a Novel Lantibiotic for the Treatment ofClostridium difficile-Associated Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01904-18 ◽

2019 ◽

Vol 63 (4) ◽

Cited By ~ 3

Author(s):

Mark E. Pulse ◽

William J. Weiss ◽

Johan A. Kers ◽

Anthony W. DeFusco ◽

Jae H. Park ◽

...

Keyword(s):

Clostridium Difficile ◽

Dose Range ◽

Hamster Model ◽

Ofclostridium Difficile ◽

Golden Syrian Hamster ◽

Content Type ◽

Lead Compounds ◽

Novel Antibiotics

ABSTRACTLantibiotics present an attractive scaffold for the development of novel antibiotics. We report here a novel lantibiotic for the treatment ofClostridium difficileinfection. The lead compounds were selected from a library of over 700 single- and multiple-substitution variants of the lantibiotic mutacin 1140 (MU1140). The best performersin vitroandin vivowere further used to challenge Golden Syrian hamsters orally in a Golden Syrian hamster model ofClostridium difficile-associated disease (CDAD) in a dose-response format, resulting in the selection of OG716 as the lead compound. This lantibiotic was characterized by a 50% effective dose of 23.85 mg/kg of body weight/day (10.97 μmol/kg/day) in this model. Upon oral administration of the maximum feasible dose (≥1,918 mg/kg/day), no observable toxicities or side effects were noted, and no effect on intestinal motility was observed. Compartmentalization to the gastrointestinal tract was confirmed. MU1140-derived variants offer a large pipeline for the development of novel antibiotics for the treatment of several indications and are particularly attractive considering their novel mechanism of action. Based on the currently available data, OG716 has an acceptable profile for further development for the treatment of CDAD.

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A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model

Clinical and Vaccine Immunology ◽

10.1128/cvi.00763-14 ◽

2015 ◽

Vol 22 (7) ◽

pp. 711-725 ◽

Cited By ~ 22

Author(s):

Natalie G. Anosova ◽

Leah E. Cole ◽

Lu Li ◽

Jinrong Zhang ◽

Anna M. Brown ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Clostridium Difficile ◽

Toxin A ◽

Healthy Human ◽

Hamster Model ◽

Toxin B ◽

Content Type ◽

Highly Virulent

ABSTRACTClostridium difficileinfection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and Bin vitroand because of theirin vivoefficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains ofC. difficiletoxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

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Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

Infection and Immunity ◽

10.1128/iai.00959-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 15

Author(s):

Jonathan L. Portman ◽

Qiongying Huang ◽

Michelle L. Reniere ◽

Anthony T. Iavarone ◽

Daniel A. Portnoy

Keyword(s):

Protein Function ◽

Inhibitory Effect ◽

Cysteine Residue ◽

Infection Model ◽

Listeriolysin O ◽

Content Type ◽

Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

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Molecular Analysis of the gyrA and gyrB Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant Clostridium difficile Mutants Selected In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01252-08 ◽

2009 ◽

Vol 53 (6) ◽

pp. 2463-2468 ◽

Cited By ~ 29

Author(s):

Patrizia Spigaglia ◽

Fabrizio Barbanti ◽

Thomas Louie ◽

Frédéric Barbut ◽

Paola Mastrantonio

Keyword(s):

Clostridium Difficile ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Toxin B ◽

Relevant Role ◽

Vitro Development ◽

Selection Of

ABSTRACT Recent studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of Clostridium difficile infections and that the acquisition of resistance to the newer fluoroquinolones is the major reason facilitating wide dissemination. In particular, moxifloxacin (MX) and levofloxacin (LE) have been recently associated with outbreaks caused by the C. difficile toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 strain. In this study, we evaluated the potential of MX and LE in the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations. Resistant mutants were obtained from five C. difficile parent strains, susceptible to MX, LE, and gatifloxacin (GA) and belonging to different toxinotypes, by selection in the presence of increasing concentrations of MX and LE. Stable mutants showing substitutions in GyrA and/or GyrB were obtained from the parent strains after selection by both antibiotics. Mutants had MICs ranging from 8 to 128 μg/ml for MX, from 8 to 256 μg/ml for LE, and from 1.5 to ≥32 μg/ml for GA. The frequency of mutation ranged from 3.8 × 10−6 to 6.6 × 10−5 for MX and from 1.0 × 10−6 to 2.4 × 10−5 for LE. In total, six different substitutions in GyrA and five in GyrB were observed in this study. The majority of these substitutions has already been described for clinical isolates or has occurred at positions known to be involved in fluoroquinolone resistance. In particular, the substitution Thr82 to Ile in GyrA, the most common found in resistant C. difficile clinical isolates, was observed after selection with LE, whereas the substitution Asp426 to Val in GyrB, recently described in toxin A-negative/toxin B-positive epidemic strains, was observed after selection with MX. Interestingly, a reduced susceptibility to fluoroquinolones was observed in colonies isolated after the first and second steps of selection by both MX and LE, with no substitution in GyrA or GyrB. The results suggest a relevant role of fluoroquinolones in the emergence and selection of fluoroquinolone-resistant C. difficile strains also in vivo.

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In VitroandIn VivoActivities of the Novel Ketolide RBx 14255 against Clostridium difficile

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00015-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5986-5989 ◽

Cited By ~ 5

Author(s):

Manoj Kumar ◽

Tarun Mathur ◽

Tarani K. Barman ◽

G. Ramkumar ◽

Ashish Bhati ◽

...

Keyword(s):

Clostridium Difficile ◽

Time Dependent ◽

Kinetics Study ◽

The Novel ◽

Promising Candidate ◽

Hamster Model ◽

Bacterial Killing ◽

Content Type ◽

Time Kill

ABSTRACTThe MIC90of RBx 14255, a novel ketolide, againstClostridium difficilewas 4 μg/ml (MIC range, 0.125 to 8 μg/ml), and this drug was found to be more potent than comparator drugs. Anin vitrotime-kill kinetics study of RBx 14255 showed time-dependent bacterial killing forC. difficile. Furthermore, in the hamster model ofC. difficileinfection, RBx 14255 demonstrated greater efficacy than metronidazole and vancomycin, making it a promising candidate forC. difficiletreatment.

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Fimbria-Encoding GeneyadCHas a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

Infection and Immunity ◽

10.1128/iai.01138-15 ◽

2015 ◽

Vol 84 (1) ◽

pp. 187-193 ◽

Cited By ~ 12

Author(s):

Renu Verma ◽

Thaís Cabrera Galvão Rojas ◽

Renato Pariz Maluta ◽

Janaína Luisa Leite ◽

Livia Pilatti Mendes da Silva ◽

...

Keyword(s):

Escherichia Coli ◽

Soft Agar ◽

Pleiotropic Effect ◽

Biological Characteristics ◽

Wild Type ◽

Content Type ◽

Pathogenic Escherichia Coli

The extraintestinal pathogen termed avian pathogenicEscherichia coli(APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07.In vitro, the transcription level ofyadCwas upregulated at 41°C and downregulated at 22°C. TheyadCexpressionin vivowas more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadCstrain presented a slightly decreased ability to adhere to HeLa cells with or without thed-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed thatfimHwas downregulated (P< 0.05) andcsgAandecpAwere slightly upregulated in the mutant strain, showing thatyadCmodulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadCstrain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P< 0.05). Motility assays in soft agar demonstrated that the ΔyadCstrain was less motile than the wild type (P< 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadCstrain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

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